REPLICATION PROTEIN-A INDUCES THE UNWINDING OF LONG DOUBLE-STRANDED DNA REGIONS

We have investigated nucleoprotein filaments composed of human replica tion protein A (RPA) and DNA by electron microscopy. At low ionic stre ngths, RPA complexes with single-stranded DNA are similar in length to protein-free DNA suggesting that RPA-bound DNA remains in an extended configuration under these conditions. However, severe compaction of R PA-DNA complexes occurs in buffers with >2 mM MgCl2 or with 100 mM NaC l. At low ionic strengths, RPA binds to A + T-rich internal regions of linear double-stranded simian virus 40 (SV40) DNA and induces separat ion of complementary DNA strands. RPA also binds to closed-circular SV 40 DNA, but requires the function of a DNA topoisomerase to invade and completely unwind duplex DNA regions. The ability of RPA to unwind lo ng stretches of double-stranded DNA is not shared by the bacterial sin gle-strand binding protein and the phage T4 gene 32 protein. (C) 1996 Academic Press Limited.